Method for induction of a heat shock protein, anti-stress or regulation of an autonomic nerve

ABSTRACT

A method for induction of a heat shock protein, anti-stress or regulation of an autonomic nerve comprises: administering a mammalian subject in need thereof a composition comprising an effective amount of hydroxymethylfurfural derivative of the following formula: 
     
       
         
         
             
             
         
       
         
         
           
             wherein R is selected from the group consisting of formula (I): 
           
         
       
    
     
       
         
         
             
             
         
       
     
     (II): HOOCCH 2 COCO—, (III): HOOCCH 2 CH 2 COCO—, and (IV): a hydrogen atom.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14366276, which entered U.S. national phase on Jun. 18, 2014 frominternational patent application No. PCT/JP2012/083040, which was filedon Dec. 20, 2012, which claimed priority from Japanese Application No.:2011-277926 filed on Dec. 20, 2011, the entire content of which isherein incorporated as reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 24, 2014, isnamed 12F096-PCT_ST25.txt and is 1,458 bytes in size.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present disclosure relates to hydroxymethylfurfural derivatives,pharmaceuticals, heat shock protein inducers, anti-stress agents,autonomic nerve regulators, foods and drinks, and methods of producingthe hydroxymethylfurfural derivative.

2. Description of the Related Art

People nowadays are exposed to long working hours, various physical andmental stress environment, or the like; and many of those peoplecomplain physical symptoms such as loss of appetite, sleep disruption,dizziness, or cold sweat, or mental symptoms such as a hatred ordistrust of other people, emotional instability, state of beingirritated, or depressed mood, even though no abnormalities are foundwhen examinations such as physical checkup. Symptoms associated withsuch unidentified complaints are often diagnosed as dysautonomia. In thepresent circumstances, such a dysautonomia is usually treated with adrug therapy such as a minor tranquilizer or hormonal agent, treatmentby diet, lifestyle changes for the better such as exercise, or the like.

It has been known that autonomic disorders including dysautonomiadescribed above are induced by excessive loads of stress. In theautonomic disorder, disturbance in the balance between the sympatheticnerve and the parasympathetic nerve (the balance of the autonomicnerve), lowered activity of the autonomic nerve, or the like isobserved. The disturbance in the balance of the autonomic nerve means astate with increased activity of the sympathetic nerve or a state withincreased activity of the parasympathetic nerve. Further, it has beenknown that the capacity to deal with stress is decreased by a loweredactivity of the autonomic nerve. For instance, because functions of thegastrointestinal tract are mainly innervated by the parasympatheticnerve, prolonged tonus of the sympathetic nerve due to loads of stresssuppresses the function of the gastrointestinal tract, resulting ingastrointestinal disorders such as loss of appetite or constipation. Inaddition, it is thought that when the parasympathetic nerve does notfunction well because of loads of stress and the activity of thesympathetic nerve remains elevated, sleep disruption is brought about.

While there is, as described above, a close relationship betweenstresses and autonomic disorders, there are also autonomic disordersthat are not caused by loads of stress. In addition, the load of stressdoes not necessarily bring about the autonomic disorder and may in somecases induce other physical symptoms.

As one of the proteins called stress proteins, there are heat shockproteins (hereinafter, referred to as HSPs). HSPs are proteins with amolecular weight of about several tens of thousands to one hundred fiftythousand and are divided into several families on the basis of themolecular weight thereof (HSP 10, HSP27, HSP40, HSP60, HSP70, HSP90, HSP110, and the like). HSPs are a group of proteins that are induced withincells when living organisms are put under physical, chemical,physiological, or mental stress. To be specific, HSPs have a role ofprotecting cells, exhibiting increased expression when living organismare exposed to various conditions including heat, bacterial infection,inflammation, reactive oxygen species, ultraviolet rays, starvation, andhypoxia. In addition, HSPs also have functions as molecular chaperonesincluding control of protein folding and inhibition of aggregation ofabnormal proteins.

Among HSPs, HSP70 has been actively studied in particular, and has beenreported to be constitutively expressed in many internal organsincluding the gastrointestinal tract and skin. Recently, HSP70'santiapoptotic action and anti-inflammatory action have been recognizedand HSP70's cell protection effect against various stresses has beenreported (Non Patent Literatures 1 to 4). Due to this, studies have cometo be conducted, attempting to apply materials with HSP70 inducingactivities in pharmaceuticals, cosmetics, or the like. When it comes tonatural product-derived materials with the HSP70 inducing activity,paeoniflorin which is a major component of Paeoniae radix has beenreported (Non Patent Literature 5).

Asparagus is a vegetable that is cultivated and harvested in variousregions including Hokkaido in Japan. It has been found that asparagushas various biological activities. Patent Literature 1 describes that anasparagus stem extract has preventive and restorative effects forvarious types of fatigue (such as physical fatigue or fatigue due tomental stress). Further, Patent Literature 2 describes that an asparagusstem extract has an effect of improving brain functions. Further, PatentLiterature 3 describes that an asparagus pseudo leaf extract has aneffect of controlling the autonomic nerve.

CITED LITERATURE Patent Literature

Patent Literature 1: Unexamined Japanese Patent Application KokaiPublication No. 2007-45750

Patent Literature 2: Unexamined Japanese Patent Application KokaiPublication No. 2007-230870

Patent Literature 3: Unexamined Japanese Patent Application KokaiPublication No. 2011-153125

Non Patent Literature

Non Patent Literature 1: Xiao-Rong Chang et al, World J Gastroenterol;13(32):4355-4359 (2007)

Non Patent Literature 2: Sarah M.et al, FASEB J. 22, 3836-3845 (2008)

Non Patent Literature 3: Hirata I et al, Digestion; 79(4):243-50 (2009)

Non Patent Literature 4: Tadashi Nishida et al, Journal of clinicalbiochemistry and nutrition; 46(1):43-51 (2010)

Non Patent Literature 5: Dai Yan et al, Cell Stress & Chaperones; 9(4),378-389 (2004)

SUMMARY OF THE INVENTION

However, there has thus far been no report concerning components inasparagus extracts or products obtained by processing asparagus, whichcomponents are involved in an anti-stress effect and autonomic nerveregulatory effect.

The present inventors discovered a novel hydroxymethylfurfuralderivative that was derived from a product obtained by heat-treatingasparagus with hot water, and found out that such ahydroxymethylfurfural derivative has an excellent HSP inducing activity,anti-stress effect, and autonomic nerve regulatory effect, therebycompleting the present disclosure. An objective of the disclosure is toprovide a novel hydroxymethylfurfural derivative, a highly effectivepharmaceutical, HSP inducer, anti-stress agent, and autonomic nerveregulator. Further, it is an objective to provide foods and drinkshaving an excellent HSP inducing activity, anti-stress effect, andautonomic nerve regulatory effect. Further, it is an objective toprovide a method of producing a hydroxymethylfurfural derivative thatcan reduce the cost and is simple and convenient.

In order to attain the above object, a hydroxymethylfurfural derivativein accordance with a first viewpoint of the present disclosure isrepresented by the general formula

(wherein, R is selected from the group consisting of the followingformula (I),

(II) HOOCCH₂COCO—, (III) HOOCCH₂CH₂COCO—, and (IV) a hydrogen atom).

The above-mentioned hydroxymethylfurfural derivative may be obtained byheat-treating an asparagus stem with hot water.

A pharmaceutical in accordance with a second viewpoint of the presentdisclosure has the above-mentioned hydroxymethylfurfural derivative asan active component.

A heat shock protein inducer in accordance with a third viewpoint of thepresent disclosure has the above-mentioned hydroxymethylfurfuralderivative as an active component.

An anti-stress agent in accordance with a fourth viewpoint of thepresent disclosure has the above-mentioned hydroxymethylfurfuralderivative as an active component.

An autonomic nerve regulator in accordance with a fifth viewpoint of thepresent disclosure has the above-mentioned hydroxymethylfurfuralderivative as an active component.

A food or drink in accordance with a sixth viewpoint of the presentdisclosure is characterized by comprising the above-mentioned heat shockprotein inducer, the above-mentioned anti-stress agent, or theabove-mentioned autonomic nerve regulator.

A method of producing a hydroxymethylfurfural derivative in accordancewith a seventh viewpoint of the present disclosure comprises the step ofheat-treating an asparagus stem with hot water.

The above-mentioned method of production may comprise the step of anenzyme treatment.

A heat shock protein inducer in accordance with an eighth viewpoint ofthe present disclosure has a product obtained by heat-treating anasparagus stem with hot water as an active component.

An anti-stress agent in accordance with a ninth viewpoint of the presentdisclosure has a product obtained by heat-treating an asparagus stemwith hot water as an active component.

An autonomic nerve regulator in accordance with a tenth viewpoint of thepresent disclosure has a product obtained by heat-treating an asparagusstem with hot water as an active component.

According to the present disclosure, a novel hydroxymethylfurfuralderivative, a highly effective pharmaceutical, HSP inducer, anti-stressagent, and autonomic nerve regulator can be provided. In addition, foodsand drinks having an excellent HSP inducing activity, anti-stresseffect, and autonomic nerve regulatory effect can be provided. Further,a method of producing a hydroxymethylfurfural derivative that can reducethe cost and is simple and convenient can be provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a figure showing the HSP70 mRNA expression inducing activityby a hydroxymethylfurfural derivative;

FIG. 2 is a figure showing the HSP70 mRNA expression inducing activitiesby hydroxymethylfurfural, a product obtained by heat-treating anasparagus stem with hot water, and a product obtained by heat-treatingan asparagus stem with hot water and enzyme treatment;

FIG. 3 is a figure showing the HSP70 protein expression inducingactivities by hydroxymethylfurfural, a product obtained by heat-treatingan asparagus stem with hot water, and a product obtained byheat-treating an asparagus stem with hot water and enzyme treatment;

FIG. 4 is a figure showing changes in lipid peroxide levels in the bloodserum by administration of a product obtained by heat-treating anasparagus stem with hot water in a mouse model of sleep deprivation;

FIG. 5 is a figure showing changes in corticosterone concentration inthe blood by administration of a product obtained by heat-treating anasparagus stem with hot water in a mouse model of sleep deprivation;

FIG. 6 is a figure showing changes in an incidence rate of hair loss byadministration of a product obtained by heat-treating an asparagus stemwith hot water in a mouse model of sleep deprivation;

FIG. 7 is a figure showing changes in the expression level of HSP70protein by administration of a product obtained by heat-treating anasparagus stem with hot water in the stomach of a mouse model of sleepdeprivation;

FIG. 8 is a figure showing changes in the expression level of HSP70protein by administration of a product obtained by heat-treating anasparagus stem with hot water in the liver of a mouse model of sleepdeprivation;

FIG. 9 is a figure showing changes in the expression level of HSP70protein by administration of a product obtained by heat-treating anasparagus stem with hot water in the kidney of a mouse model of sleepdeprivation;

FIG. 10 is a figure showing changes in the expression level of HSP70mRNA by administration of a product obtained by heat-treating anasparagus stem with hot water in human;

FIG. 11 is a figure showing changes in autonomic nervous balance byadministration of a product obtained by heat-treating an asparagus stemwith hot water in human;

FIG. 12 is a figure showing changes in autonomic nervous activity byadministration of a product obtained by heat-treating an asparagus stemwith hot water in human;

FIG. 13 is a figure showing changes in the expression level of HSP70mRNA by administration of a product obtained by heat-treating anasparagus stem with hot water and enzyme treatment in human;

FIG. 14 is a figure showing changes in autonomic nervous balance byadministration of a product obtained by heat-treating an asparagus stemwith hot water and enzyme treatment in human; and

FIG. 15 is a figure showing changes in an autonomic nervous activity byadministration of a product obtained by heat-treating an asparagus stemwith hot water and enzyme treatment in human.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

Embodiments of the present disclosure will be described in detail below.

(1. Hydroxymethylfurfural Derivative)

The hydroxymethylfurfural derivative according to the present disclosureis represented by the following general formula.

In the above general formula, R selected from the group consisting ofthe following formula (I),

(II) HOOCCH₂COCO—, and (III) HOOCCH₂CH₂COCO—, and (IV) a hydrogen atom.

In cases where R is the above formula (I), the hydroxymethylfurfuralderivative is represented by the following structural formula.

In cases where R is the above formula (I), the hydroxymethylfurfuralderivative includes the following two stereo isomers (R form and Sform).

In cases where R is the above (IV) a hydrogen atom, thehydroxymethylfurfural derivative is represented by the followingstructural formula (name of compound: hydroxymethylfurfural).

The hydroxymethylfurfural derivative according to the present disclosuremay be, as shown below, obtained by heat-treating an asparagus stem withhot water.

The hydroxymethylfurfural derivative according to the presentdisclosure, as shown below, has an excellent heat shock protein inducingactivity, anti-stress effect, and autonomic nerve regulatory effect.

(2. Method of Producing a Hydroxymethylfurfural Derivative)

The method of producing a hydroxymethylfurfural derivative according tothe present disclosure comprises the step of heat-treating an asparagusstem with hot water.

In the present specification, the phrase “heat-treating with hot water”means heat-treating in hot water. As an asparagus stem used in thepresent disclosure, the stem portion of, for example, green asparagus,white asparagus, purple asparagus, or the like can be used. Further, theorigin of asparagus is not particularly restricted; and asparagus thatis domestically produced may be used or asparagus that is imported maybe used. As long as asparagus is one that brings out effects of thepresent disclosure, asparagus may be selected as appropriate.

The step of heat-treating an asparagus stem with hot water is carriedout by, for example, adding 1 to 50 volumes of water to an asparagusstem and heating in hot water for 20 to 180 minutes. A temperature onthis occasion is, preferably, 50 to 300° C. In cases where the heattreatment is carried out under atmospheric pressure, it is preferred tobe, for example, a temperature of 100° C., or more. It is to be notedthat the heat treatment with hot water may be carried out under pressurebeing applied and the pressure is preferably, for example, 0.1 to 0.2MPa (for example, 0.12 MPa in cases where an autoclave is used).

As described above, the hydroxymethylfurfural derivative according tothe present disclosure is obtained by heat-treating an asparagus stemwith hot water. Thus, the method of producing a hydroxymethylfurfuralderivative according to the present disclosure comprises the step ofheat-treating an asparagus stem with hot water. In such a method ofproduction, because the stem portion of asparagus which is widelydistributed as a vegetable is used, the hydroxymethylfurfural derivativecan be produced at low cost. In addition, the hydroxymethylfurfuralderivative can be simply and conveniently produced by heat-treatingasparagus with hot water without using sophisticated techniques, specialdevices, or the like. Further, because asparagus which is a foodmaterial is subjected to the heat treatment with hot water, thehydroxymethylfurfural derivative obtained by such a method of productioncan be said to be high in safety; and the asparagus stem can besterilized because of heating with hot water. It is to be noted that aslong as a method of heat treatment with hot water is one that brings outeffects of the present disclosure, the method may be selected asappropriate.

For the purpose of increasing the efficiency of the step of heattreatment with hot water to efficiently produce hydroxymethylfurfuralderivatives, the method of producing a hydroxymethylfurfural derivativeaccording to the present disclosure may comprise additional stepsillustrated below.

Examples of the above additional step may include the step of finelychopping asparagus stems before the heat treatment with hot water. Theasparagus stem can finely chopped into pieces of about 0.5 to 10 cm insize. The fine chopping may be manually carried out using, for example,a knife, cutter, or the like. Or a machine such as a chopping machine ormill may be used. As long as a method of finely chopping is one thatbrings out effects of the present disclosure, the method may be selectedas appropriate.

Examples of the above additional step may include the step ofcompressing asparagus stems before the heat treatment with hot water.The asparagus stem can be compressed using, for example, a compressor.As long as a method of compressing is one that brings out effects of thepresent disclosure, the method may be selected as appropriate.

Examples of the above additional step include, for the purpose ofbreaking down plant tissues or the like, the step of an enzyme treatmentbefore or after the step of the heat treatment with hot water. Theenzyme treatment increases the efficiency of the step of the heattreatment with hot water, which allows hydroxymethylfurfural derivativesto be more efficiently produced. For instance, an enzyme such ascellulase, hemicellulase, pectinase, amylase, or pullulanase; or acombination of 2, 3, or more of these enzymes is suitably used for thepurpose of efficiently breaking down fibers, pectin, or the like in theasparagus stem. As long as an enzyme is one that brings out effects ofthe present disclosure, the enzyme may be selected as appropriate. Inthe step of the enzyme treatment, an amount to be added, temperature,and reaction time that are most appropriate for the enzyme to be usedmay be selected. In cases where cellulase is used, the enzyme treatmentcan be carried out, for example, at an amount of cellulase added of 0.1to 5% (w/w) at a temperature of 30 to 60° C., for 1 to 72 hours. Thestep of the enzyme treatment may be carried out before the step of theheat treatment with hot water or may be after the step of the heattreatment with hot water. It is to be noted that, from the viewpoint ofefficiently breaking down cellulose in the asparagus stem to moreefficiently produce the hydroxymethylfurfural derivative, it ispreferred to carry out the enzyme treatment by cellulase after the stepof the heat treatment with hot water. As long as a method of enzymetreatment is one that brings out effects of the present disclosure, themethod may be selected as appropriate.

Examples of the above additional step include the step of mechanicallygrinding residues after the heat treatment with hot water. In thegrinding, a machine such as a mill or blender may, for example, be used.As long as a method of grinding is one that brings out effects of thepresent disclosure, the method may be selected as appropriate.

Examples of the above additional step include the step of centrifugationor filtration after the heat treatment with hot water. Further inclusionof these steps enables the residue to be efficiently removed to obtain aheat treatment liquid. The centrifugation may be carried out, forexample, at a revolution of 3,000 to 7,000 rpm at 4 to 50° C. In thefiltration, a commercially available filter paper, filter cloth, or thelike may, for example, be used. As long as a method of centrifugation ora method of filtration is one that brings out effects of the presentdisclosure, the method may be selected as appropriate.

Examples of the above additional step include the step of concentratingthe obtained heat treatment liquid under reduced pressure after the heattreatment with hot water. The concentration can be carried out, forexample, by an evaporator or the like. As long as a method ofconcentration is one that brings out effects of the present disclosure,the method may be selected as appropriate.

Examples of the above additional step include the step of spray dryingor freeze drying the heat treatment liquid after the heat treatment withhot water. The spray drying may be carried out, for example, at anexhaust air temperature of 70 to 90° C., and a chamber temperature of 80to 100° C. As long as a method of spray drying or a method of freezedrying is one that brings out effects of the present disclosure, themethod may be selected as appropriate.

By further including the additional step illustrated above in the stepof heat-treating an asparagus stem with hot water, thehydroxymethylfurfural derivative can be more efficiently produced. Aslong as an additional step is one that brings out effects of the presentdisclosure, the step may be selected as appropriate.

The step of heat-treating an asparagus stem with hot water will beillustrated below. The asparagus stem is finely chopped into pieces ofabout 0.5 to 10 cm and added with 1 to 50 volumes of water. The heattreatment with hot water is carried out at 50 to 100° C., or at 121° C.under pressure being applied, for 20 to 180 minutes. The resultant was,after allowed to cool, added with cellulase at 0.1 to 5% (w/w); andenzyme treatment is carried out at 30 to 60° C., for 1 to 72 hours.Subsequently, residues are then mechanically ground and centrifuged at arevolution of 3,000 to 7,000 rpm at 4 to 50° C., to obtain asupernatant. Such a supernatant is then subjected to spray drying at anexhaust air temperature of 70 to 90° C., and a chamber temperature of 80to 100° C.

In the present specification, a “product obtained by heat-treating anasparagus stem with hot water” refers to one obtained by heat-treatingan asparagus stem in hot water, and then removing residues bycentrifugation, filtration, or the like, followed by concentration.Further, in the present specification, a “product obtained byheat-treating an asparagus stem with hot water and an enzyme treatment”means a product obtained by heat-treating an asparagus stem with hotwater that is obtained by going through the step of the enzyme treatmentas described above before or after the step of the heat treatment withhot water. In the product obtained by heat-treating an asparagus stemwith hot water and the product obtained by heat-treating an asparagusstem with hot water and enzyme treatment, the aforementionedhydroxymethylfurfural derivative is contained, for example, at least0.05% or more as an active component.

The obtained hydroxymethylfurfural derivative by the method ofproduction according to the present disclosure can be fractionated by,for example, dissolving the product obtained by heat-treating anasparagus stem with hot water in water or an organic solvent (such asmethanol) and subjecting to open column chromatography with a reversephase carrier (for example, DIAION HP-20 (product name) (manufactured byMitsubishi Chemical Corporation) or the like). It also can, for example,be fractionated by a carrier for gel filtration (for example, SephadexLH-20 (product name) (manufactured by Pharmacia Fine Chemicals) or thelike). Further, a predetermined fraction that is eluted by theabove-mentioned method can be purified, for example, by isolating byhigh performance liquid chromatography (HPLC).

Without wishing to be bound by a particular theory, it is thought that,by heat-treating the asparagus stem with hot water as described above,an organic acid and sugars derived from the asparagus stem are reactedat high temperatures, thereby obtaining the hydroxymethylfurfuralderivative according to the present disclosure. Examples of the organicacid include pyroglutamic acid, α-ketoglutaric acid, and oxaloaceticacid. Meanwhile, examples of the sugar include fructose, glucose,sucrose, or mannose.

For instance, it is thought that, in the step of heat-treating anasparagus stem with hot water, pyroglutamic acid and fructose which arederived from the asparagus stem are reacted at high temperatures,thereby obtaining the following compound.

In the method of producing a hydroxymethylfurfural derivative accordingto the present disclosure, plants other than asparagus, the plantcontaining an organic acid and sugar illustrated above, can be used asappropriate. Vegetables that contain pyroglutamic acid and fructose can,for example, preferably be used. Vegetables such as cabbage, broccoli,pumpkin, onion, garlic, or carrot may, for example, be suitably used. Aslong as a plant is one that brings out effects of the presentdisclosure, the plant may be selected as appropriate.

(3. HSP Inducer, Anti-Stress Agent, and Autonomic Nerve Regulator)

By the present disclosure, an HSP inducer, anti-stress agent, andautonomic nerve regulator that contain the hydroxymethylfurfuralderivative according to the present disclosure as an active componentare provided.

The HSP inducer according to the present disclosure may be used in orderto induce HSP that is present in vivo or in vitro. HSP used here is, forexample, HSP70, HSP10, HSP27, HSP40, HSP60, HSP90, HSP 110, or the like,with HSP70 being preferred. An HSP inducing activity can be evaluated,for example, by culturing cells with such a HSP inducer being added andmeasuring, by a known method, an HSP mRNA expression inducing activity,HSP protein expression inducing activity, or the like. As long as amethod of evaluation is one that brings out effects of the presentdisclosure, the method may be selected as appropriate.

The anti-stress agent according to the present disclosure can beadministered to a living organism to thereby obtain an anti-stresseffect. The anti-stress effect can be evaluated, for example, byadministrating such an anti-stress agent to mammals and measuring anoxidative stress index, stress hormone concentration in the blood, orthe like before and after the administration. As long as a method ofevaluation is one that brings out effects of the present disclosure, themethod may be selected as appropriate.

The autonomic nerve regulator according to the present disclosure can beadministered to living organisms to thereby obtain an autonomic nerveregulatory effect. The autonomic nerve regulatory effect can beevaluated, for example, by administrating such an autonomic nerveregulator to mammals and measuring an autonomic nervous balance, anautonomic nervous activity, or the like before and after theadministration. As long as it is a method of evaluation is one thatbrings out effects of the present disclosure, the method may be selectedas appropriate.

Further, by the present disclosure, an HSP inducer, anti-stress agent,and autonomic nerve regulator that contain a product obtained byheat-treating an asparagus stem with hot water as an active componentare also provided. Such a product obtained by heat-treating an asparagusstem with hot water is, as described above, obtained by heat-treating anasparagus stem with hot water. Therefore, the hydroxymethylfurfuralderivative according to the present disclosure is contained in such aproduct obtained by heat-treating an asparagus stem with hot water.

(4. Foods and Drinks, and Pharmaceuticals)

By the present disclosure, foods and drinks that contain the HSPinducer, anti-stress agent, and autonomic nerve regulator according tothe present disclosure are provided. Such foods and drinks can beprocessed, by a conventional method, into a form suitable to eat anddrink including, for example, a form of granule, a form of particulate,tablets, capsules, a form of gel, a form of cream, a form of paste, aform of suspension, a form of aqueous solution, a form of emulsion, anda form of powder. In addition, excipients, binders, lubricants, coloringagents, disintegrants, thickeners, preservatives, stabilizers, pHadjusters, or the like, which are usually used in foods and drinks, canbe added. Further, for the purpose of improving the quality of taste,sugars, sugar alcohols, salts, fats and oils, amino acids, organicacids, glycerin, or the like can be added in a range where the effectsof the present disclosure is not impaired. It is to be noted that incases where the HSP inducer, anti-stress agent, and autonomic nerveregulator according to the present disclosure are added to existingfoods and drinks and used, any foods and drinks may be selected asappropriate as base foods and drinks as long as the foods and drinks areones that bring out effects of the present disclosure.

In cases where the HSP inducer, anti-stress agent, and autonomic nerveregulator according to the present disclosure are used as a food ordrink, the food or drink can be taken at, for example, 50 mg to 2000mg/day, and preferably 100 mg to 1000 mg/day, in terms of a productobtained by heat-treating an asparagus stem with hot water (or a productobtained by heat-treating an asparagus stem with hot water and enzymetreatment) to thereby obtain a desired HSP inducing activity,anti-stress effect, and autonomic nerve regulatory effect. The intakeamount taken may be selected as appropriate on the basis of an object ofintake, a form of food or drink, or the like.

The foods and drinks according to the present disclosure have both ananti-stress effect and autonomic nerve regulatory effect. Therefore, itis expected that the anti-stress effect and autonomic nerve regulatoryeffect synergistically act to bring out a higher effect on autonomicdisorders by loads of stress. In addition, it is expected to bring outan autonomic nerve regulatory effect also for autonomic disorders thatare not caused by loads of stress.

The HSP inducer, anti-stress agent, and autonomic nerve regulatoraccording to the present disclosure can be used as a pharmaceutical. Thepharmaceutical according to the present disclosure contains theaforementioned hydroxymethylfurfural derivative as an active component.In this case, the pharmaceutical can be prepared, by a conventionalmethod, in a dosage form including, for example, tablets, granules,powders, capsules, syrups, and injection solutions. Further, excipients,binders, lubricants, coloring agents, disintegrants, thickeners,preservatives, stabilizers, pH adjusters, or the like, which are usuallyused in pharmaceuticals, can be added. A method of administration may beselected as appropriate in a range where effects of the presentdisclosure is brought out, which method includes oral administration,intravenous administration, intraperitoneal administration, intradermaladministration, and sublingual administration.

In cases where the HSP inducer, anti-stress agent, and autonomic nerveregulator according to the present disclosure are used as apharmaceutical, the pharmaceutical can be administrated at, for example,50 mg to 2000 mg/day, and preferably 100 mg to 1000 mg/day, in terms ofa product obtained by heat-treating an asparagus stem with hot water (ora product obtained by heat-treating an asparagus stem with hot water andenzyme treatment) to thereby obtain a desired HSP inducing activity,anti-stress effect, and autonomic nerve regulatory effect. The dosagemay be selected as appropriate on the basis of an object ofadministration, dosage form, patients' age and body weight, or the like.

EXAMPLES

By way of the examples, the present disclosure will be concretelydescribed below. However, the present disclosure is by no means limitedto these examples.

Example 1

(Production of Hydroxymethylfurfural by Heat-Treating an Asparagus Stemwith Hot Water)

Water 1.5 L was added to green asparagus stem (fresh weight 1.5 kg); andthe mixture was, for the purpose of heat treatment with hot water,autoclaved (121° C., for 20 minutes) using an autoclave and filteredwith a filter cloth. The obtained liquid was concentrated under reducedpressure in an evaporator to obtain a heat treatment product. Theobtained heat treatment product was fractionated by columnchromatography (product name: DIAION HP-20, manufactured by MitsubishiChemical Corporation; 500 mL, elution; H₂O, 50% methanol, 100% methanol)to obtain 1.7 g. Next, the obtained fractionation product was purifiedby preparative HPLC (product name: Hitachi L-7100, manufactured byHitachi Ltd.) to obtain the compound (X) (5.0 mg). A column whoseproduct name: CAPCELL PAK C18 UG 120, 20φ×250 mm (manufactured byShiseido Co., Ltd.) was used; and the mobile phase was as shown in Table1 (A: H₂O, B: methanol). The flow rate of preparative HPLC was 8 mL/minand detection was carried out at a detection wavelength of 280 nm by anultraviolet absorbance detector.

TABLE 1 Time (minutes) A(%) B(%) 0 80 20 10 80 20 30 50 50 40 50 50 4180 20 45 80 20

The NMR data of the compound (X) obtained as described above are shownbelow.

¹H-NMR (400 MHz, DMSO-d₆)

δ 4.49 (1H,dd,J=5.2Hz)

5.59 (1H,dt,J=5.6 Hz)

6.55 (1H,d,J=3.6 Hz)

7.48 (1H,d,J=3.6 Hz)

9.52 (1H,s)

¹³C-NMR (100 MHz, DMSO-d₆)

δ 55.9

109.7

124.5

151.7

162.1

178.0

In order to determine the structure of the compound (X) obtained above,the NMR data of a commercially available product ofhydroxymethylfurfural (product name: 5-Hydroxymethyl-2-furaldehyde,Tokyo Chemical Hanbai Co., Ltd.) was compared with that of the compound(X). The NMR data of the commercially available product ofhydroxymethylfurfural is shown below.

¹H-NMR (400 MHz, DMSO-d₆)

δ 5.51 (1H,dd,J=5.2 Hz)

5.59 (1H,dt,J=5.6 Hz)

6.60 (1H,d,J=3.6 Hz)

7.49 (1H,d,J=3.6 Hz)

9.56 (1H,s)

¹³C-NMR (100 MHz, DMSO-d₆)

δ55.9

109.7

124.4

151.7

162.1

177.9

Because the NMR data of the compound (X) obtained above matched that ofthe commercially available product, it was proven that the compound (X)was hydroxymethylfurfural (the following structural formula). From theabove, it became apparent that hydroxymethylfurfural was contained inthe product obtained by heat-treating an asparagus stem with hot water.

Example 2

(Production of Novel Hydroxymethylfurfural Derivative by Heat-Treatingan Asparagus Stem with Hot Water)

Water 1 L was added to green asparagus stem (fresh weight 1.0 kg); andthe mixture was, for the purpose of heat treatment with hot water,autoclaved (121° C., for 20 minutes) using an autoclave and filteredwith a filter cloth. The obtained liquid was concentrated under reducedpressure in an evaporator to obtain a product obtained by heat-treatingan asparagus stem with hot water. The obtained heat treatment productwas fractionated by column chromatography (product name: DIMON HP-20,manufactured by Mitsubishi Chemical Corporation; 500 mL, elution; H₂O,30% methanol, 100% methanol). Next, the obtained fractionation product723.8 mg was purified by column chromatography (product name: SephadexLH-20, manufactured by Pharmacia Fine Chemicals;250 mL, elution; H₂O).Further, the obtained fractionation product 12.6 mg was purified bypreparative HPLC (product name: Hitachi L-7100, manufactured by HitachiLtd.) to obtain the compound (Y) (2.0 mg). The conditions for thepreparative HPLC were the same as those in Example 1.

The compound (Y) obtained above was subjected to HPLC analysis (productname: Hitachi L-7100, manufactured by Hitachi Ltd.) and, as a result,the retention time was found to be 22.99 minutes. In this analyticalHPLC, a column whose product name: CAPCELL PAK C18 UG 120, 4.6φ×250 mm(manufactured by Shiseido Co., Ltd.) was used; and the mobile phase wasas shown in Table 2 (C: 20 mM sodium phosphate buffer (pH 2.3), D:acetonitrile). The flow rate of analytical HPLC was 1 mL/min anddetection was carried out at a detection wavelength of 280 nm by anultraviolet absorbance detector.

TABLE 2 Time (minutes) C(%) D(%) 0 95 5 5 95 5 20 90 10 25 90 10 30 5050 35 50 50 36 95 5

The LC/Tof MS analysis data of the compound (Y) obtained above are shownbelow.

Found m/z 238.0710([M+H]+); C₁₁H₁₂NO₅

Theoretical value m/z 238.0715([M+H]+); C₁₁H₁₂NO₅

From the above, it became apparent that the compound (Y) obtained abovehas a molecular formula of C₁₁H₁₁NO₅.

The ¹H-NMR data of the compound (Y) obtained above are shown below.

¹H-NMR (400 MHz, CD₃OD)

δ 2.00(4H,m)

4.20 (1H,dd,J=3.9,9.2 Hz)

5.20 (2H,s)

6.55 (1H,d,J=3.6 Hz)

7.37 (1H,d,J=3.6 Hz)

9.45 (1H,s)

Example 3

(Synthesis of Novel Hydroxymethylfurfural Derivative)

It was estimated that the compound (Y) (C₁₁H₁₁NO₅) obtained in Example 2was generated by reacting pyroglutamic acid (C₅H₇NO₃) and fructose(C₆H₁₂O₆) derived from the asparagus stem under heating. In order toverify this, a compound was synthesized by the following method usingpyroglutamic acid and fructose. It is to be noted that because stereoisomers were, as described below, thought to exist for the compound (Y),an S(L) form and R(D) form were synthesized with L-pyroglutamic acid asa starting material and with D-pyroglutamic acid as a starting material,respectively.

L-pyroglutamic acid (product name: L-pyroglutamic acid, Tokyo ChemicalHanbai Co., Ltd.) 3.0 g and D-fructose (product name: D(−)-fructose,Junsei Chemical Co., Ltd.) 1.5 g were mixed in an Erlenmeyer flask andautoclaved (121° C., for 20 minutes) using an autoclave. The obtainedreaction product was fractionated by column chromatography (productname: DIMON HP-20, manufactured by Mitsubishi Chemical Corporation; 150mL, elution; H2O, 30% methanol, 100% methanol). Further, a 100% methanolfraction was purified by preparative HPLC using a column (product name:CAPCELL PAK C18 UG 120, 20φ×250 mm, manufactured by Shiseido Co., Ltd.)to obtain S form compound (Z) (24.4 mg). The conditions for thepreparative HPLC were the same as those in Example 1.

D-pyroglutamic acid (product name: D-pyroglutamic acid, Tokyo ChemicalHanbai Co., Ltd.) 2.0 g and D-fructose (product name: D(−)-fructose,Junsei Chemical Co., Ltd.) 1.0 g were mixed in an Erlenmeyer flask andautoclaved (121° C., for 20 minutes) using an autoclave. The obtainedreaction product was fractionated by column chromatography (productname: DIMON HP-20, manufactured by Mitsubishi Chemical Corporation; 100mL, elution; H₂O, 30% methanol, 60% methanol). A 60% methanol fractionwas concentrated to about 50 mL under reduced pressure by an evaporatorand then separated with ethyl acetate (50 mL×5). An ethyl acetate layerwas concentrated under reduced pressure by an evaporator andfractionated by column chromatography (product name: DIAION HP-20,manufactured by Mitsubishi Chemical Corporation; 10 mL, elution; H₂O,30% methanol, 60% methanol). A 60% methanol fraction was concentratedunder reduced pressure by an evaporator to obtain R form compound (Z)(24.6 mg).

The S form and R form compounds (Z) obtained above were subjected toHPLC analysis (product name: Hitachi L-7100, manufactured by HitachiLtd.) and, as a result, the retention time was found to be 22.89 minutesfor each. The conditions for the analytical HPLC were the same as thosein Example 2.

With regard to the S form and R form compounds (Z) obtained above, MSdata and NMR analysis data are shown below.

EI-MS: m/z 237

EI-HR-MS: m/z 237.0612; C₁₁H₁₁NO₅

¹H-NMR (400 MHz, CD₃OD): S form

δ 2.33 (4H,m)

4.34 (1H,dd,J=3.9,9.0 Hz)

5.51 (2H,s)

6.73 (1H,d,J=3.4 Hz)

7.38 (1H,d,J=3.4 Hz)

9.57 (1H,s)

¹³C-NMR (100 MHz, CD₃OD): S form

δ 25.8

30.2

57.0

59.6

114.0

124.0

154.5

156.7

173.4

179.6

181.1

¹-NMR (400 MHz, CD₃OD): R form

δ 2.33 (4H,m)

4.34 (1H,dd,J=3.9,9.0 Hz)

5.27 (2H,s)

6.73 (1H,d,J=3.6 Hz)

7.38 (1H,d,J=3.6 Hz)

9.57 (1H,s)

¹³C-NMR (100 MHz, CD₃OD): R form

δ 25.8

30.3

57.0

59.6

113.7

124.0

154.5

156.8

173.4

179.6

181.1

In order to determine the absolute structure of the compound (Y)obtained in Example 2, the compound (Y) obtained in Example 2, the Sform compound (Z) and R form compound (Z), both of which were obtainedabove, were subjected to HPLC analysis (product name: Hitachi L-7100,manufactured by Hitachi Ltd.) using a chiral column (product name:CHIRAL PAK IA, 4.6φ×150 mm, manufactured by Daicel Corporation). Theconditions for the analytical HPLC were the same as those in Example 2except that the different column was used. As a result, the retentiontime of the S form compound (Z) was found to be 18.92 minutes and theretention time of the R form compound (Z) was found to be 20.57 minutes.Because the retention time of the compound (Y) obtained in Example 2 was19.06 minutes, it became apparent that the compound (Y) obtained inExample 2 was the S form.

When the HPLC analysis data, LC/Tof MS analysis data, and ¹H-NMR data inExample 2 were compared with the above-mentioned analysis data in thisExample, the compound (Y) obtained by Example 2 and the compound (Z)obtained by this Example were shown to be identical compounds.Therefore, it became apparent that the product obtained by heat-treatingan asparagus stem with hot water, which product was obtained in Example2, at least contained the S form of hydroxymethylfurfural derivativehaving the following structural formula:

Example 4

(Evaluation of HSP70 mRNA Expression Inducing Activity)

With regard to a commercially available product of hydroxymethylfurfural(the same as n Example 1) (hereinafter, referred to as sample 1), the Sform of hydroxymethylfurfural derivative synthesized in Example 3(hereinafter, referred to as sample 2-S), and the R form ofhydroxymethylfurfural derivative synthesized in Example 3 (hereinafter,referred to as sample 2-R), and a product obtained by heat-treating anasparagus stem with hot water, which product was produced by thefollowing method, (hereinafter, referred to as sample 3), and a productobtained by heat-treating an asparagus stem with hot water and enzymetreatment (hereinafter, referred to as sample 4), an HSP70 inducingactivity was evaluated by measuring the mRNA expression level of HSP70.

A method of producing sample 3 is shown below. To green asparagus stems(fresh weight 6.63 kg), water 28.5 L was added; and the mixture was, forthe purpose of heat treatment with hot water, autoclaved (121° C., 20minutes). After cooling, filtration was carried out with a filter paper(product name: Toyo Roshi No. 5A, manufactured by Toyo Roshi Kaisha,Ltd.); and concentration was carried out by an evaporator. To about 10 Lof the concentrated liquid, an excipient (product name: Pinedex,manufactured by Matsutani Chemical Industry Co., Ltd.) 275.4 g was addedand the resulting mixture was freeze-dried to obtain 542.4 g of powdercontaining a product obtained by heat-treating an asparagus stem withhot water (in this, the product obtained by heat-treating an asparagusstem with hot water derived from the solid content of asparagus stemaccounted for 267.0 g and the excipient accounted for 275.4 g).

A method of producing sample 4 is shown below. To green asparagus stems(fresh weight 12 kg), water 24 L was added; and the mixture was, for thepurpose of heat treatment with hot water, autoclaved (121° C., 20minutes). After allowed to cool to 45° C., the resultant was added withsucrase C (product name) (manufactured by Mitsubishi-Kagaku FoodsCorporation) 20 g and Macerozyme A (product name) (manufactured byYakult Pharmaceutical Industry Co., Ltd.) 20 g; and enzyme treatment wascarried out at 45° C., for three days. Subsequently, autoclave (121° C.,20 minutes) was carried out and filtration was carried out with a filtercloth to collect a filtrate 35 L. Concentration was carried out by anevaporator until the volume reached 9 L. To this concentrated liquid, anexcipient (product name: Pinedex, manufactured by Matsutani ChemicalIndustry Co., Ltd.) 1.20 kg was added; and the resulting mixture wasagain autoclaved (121° C., 20 minutes). Subsequently, freeze drying wascarried out to obtain 2.12 kg of a powder containing a product obtainedby heat-treating an asparagus stem with hot water and enzyme treatment(in this, the product obtained by heat-treating an asparagus stem withhot water and enzyme treatment derived from the solid content ofasparagus stem accounted for 0.92 kg and the excipient accounted for1.20 kg).

First, with regard to the sample 1, sample 2-S, and sample 2-R, an HSP70mRNA expression level was evaluated using human promyelocytic leukemiacells (HL-60 cells).

A method of evaluating the HSP70 mRNA expression level using humanpromyelocytic leukemia cells (HL-60 cells) is shown below. HL-60(source: Dainippon Pharmaceutical Co., Ltd.) was suspended in RPMI1640medium (product name: RPMI1640 medium “NISSUI” (2) powder, manufacturedby Nissui Pharmaceutical Co., Ltd.) supplemented with 10% fetal bovineserum (FBS) (product name: MultiSer, manufactured by Thermo Trace) andwas transferred into a 1.5 mL sampling tube (500,000 cells/1 mL/tube).At the same time, 0.1 mL of each of the samples (sample 1, sample 2-S,and sample 2-R) that had been prepared with ion-exchanged water wasadded such that the final concentration was 1 mg/mL. To a control,ion-exchanged water 0.1 mL was added. After cultured at 37° C., in thepresence of 5% CO₂ for four hours, cells were harvested at 3,000 rpm andsubjected to mRNA detection. From this, total RNA was extracted usingTRIzol reagent (manufactured by Life Technologies Corporation) and theconcentration thereof was measured by Nanodrop (manufactured by ThermoFisher Scientific K.K.). Using a cDNA synthesis kit (product name:ReverTra Ace qPCR RT Master Mix with gDNA Remover, manufactured byToyobo Life Science), cDNA was synthesized. A reaction liquid after thereverse transcription was diluted with Nuclease-free water so as to havea concentration of 3 ng/μL to be used as a template for real-time PCR.

In PCR, HSP70 forward primer (SEQ ID NO: 1) and HSP70 reverse primer(SEQ ID NO: 2) were used as primers. Beta 2 microglobulin gene was usedas an internal control gene for correction of the HSP70 gene expression;and beta 2 microglobulin forward primer (SEQ ID NO: 3) and beta 2microglobulin reverse primer (SEQ ID NO: 4) were used as primerstherefor. Real-time PCR was carried out using a reaction kit (productname: SsoAdvanced SYBR Green Supermix, manufactured by Bio-RadLaboratories, Inc.) by a real time PCR analysis system (product name:CFX Connect, manufactured by Bio-Rad Laboratories, Inc.). A total of 10μL of PCR reaction liquid was subjected to a three-minute incubation at95° C., (initial denaturation) followed by repetition of 40 cycles, eachcycle of which comprised denaturation at 95° C., for 1 second andannealing at 59° C., for 10 seconds.

Using Cq value obtained by the above real time PCR analysis system, aratio of expression level of HSP70 gene was calculated on the basis ofthe following calculation formula (ΔΔCt method). It is to be noted thatthe Cq value represents the number of reaction cycles at the time whenthe level of amplified gene reaches a certain predetermined level in theamplification reaction of the gene.

Cq value of control HSP70: A

Cq value of control B2M: B

Cq value of sample HSP70: C

Cq value of sample B2M: D

ΔCq(control)=A-B

ΔCq(sample)=C-D

Δ(ΔCq)=ΔCq(sample)−ΔCq(control)

Ratio of the expression level=2^(−Δ(ΔCq))

The results are shown in FIG. 1. In FIG. 1, the HSP70 mRNA expressioninducing activity of the sample 1, sample 2-S, and sample 2-R wasexpressed as a ratio (%) to that of the control. As compared with thecontrol, the samples 1, 2-S, and 2-R exhibited an about 3- to 9-foldincreased HSP70 mRNA expression (samples 2-S and 2-R, **p<0.01 vs.control; sample 1, *p=0.069 vs. control). From this, it became apparentthat hydroxymethylfurfural and the S form and R form ofhydroxymethylfurfural derivatives having the following structuralformula had the HSP70 inducing activity at an mRNA expression level.

Next, with regard to the sample 1, sample 3, and sample 4, the HSP70mRNA expression level was evaluated using human uterine cervical cancercells (HeLa cells).

Human uterine cervical cancer cells (HeLa cells) (source: Incorporatedadministrative agency RIKEN, the Institute of Physical and ChemicalResearch, BioResource Center) were suspended in Dulbecco's ModifiedEagle's Medium (DMEM) (product name: Dulbecco's Modified Eagle's Medium“Nissui” 2 powder, manufactured by Nihon Pharmaceutical Co., Ltd.)supplemented with 10% fetal bovine serum (FBS) (product name: MultiSer,manufactured by Thermo Trace) and seeded a six-well plate (200,000cells/2 mL/well). On the following day, the medium was replaced withfresh DMEM (1.8 mL); and 0.2 mL of each of the samples was added, whichsamples had been each prepared with ion-exchanged water so as to have afinal concentration of 1 mg/mL (with regard to the samples 3 and 4, afinal concentration of 1 mg/mL in terms of a product obtained byheat-treating an asparagus stem with hot water and a product obtained byheat-treating an asparagus stem with hot water and enzyme treatmentderived from the solid content of asparagus stem). To a control,ion-exchanged water 0.2 mL was added. After cultured for 22 hours, cellswere scraped off with a cell scraper and subjected to mRNA detection.From this, total RNA was extracted using an RNA extraction kit (productname: Fast Pure RNA kit, manufactured by Takara Bio Inc.), diluted 100fold with DEPC treated water; and the absorbance (wavelength 260 nm) wasmeasured by a spectrophotometer. The RNA concentration was calculatedusing the calculation formula: absorbance (wavelength 260nm)×40×dilution factor=RNA concentration (ng/μL). An RNA solution wasdiluted to an optional concentration with a TE buffer; and cDNA wassynthesized using a cDNA synthesis kit (product name: Prime Script 1stStrand cDNA synthesis kit, manufactured by Takara Bio Inc.). Oligo dTprimer (product name) (manufactured by Takara Bio Inc.) was used as aprimer. A reaction liquid after the reverse transcription was dilutedwith the TE buffer so as to have a concentration of 10 ng/μL to be usedas a template for PCR.

In PCR, HSP70 forward primer (SEQ ID NO: 5) and HSP70 reverse primer(SEQ ID NO: 6) were used as primers. Beta 2 microglobulin gene was usedas an internal control gene for correction of the HSP70 gene expression;and beta 2 microglobulin forward primer (SEQ ID NO: 3) and beta 2microglobulin reverse primer (SEQ ID NO: 4) were used as primerstherefor. For PCR, a PCR enzyme (product name: TaKaRa Ex Taq,manufactured by Takara Bio Inc.) was used. A total of 20 μL of PCRreaction liquid was subjected to a one-minute incubation at 94° C.,(initial denaturation), followed by repetition of 32 cycles (HSP70) or24 cycle (beta 2 microglobulin), each cycle of which compriseddenaturation at 94° C., for 30 seconds, annealing at 57° C., (HSP70) or59° C., (beta 2 microglobulin) for 30 seconds, and elongation at 72° C.,for 30 seconds. An elongation reaction at 72° C., for 30 seconds wasperformed to end all PCRs. The PCR reaction liquid was electrophoresedby a conventional method; and staining with ethidium bromide was carriedout.

By measuring the fluorescence intensity under ultraviolet irradiation inAlphaView (product name) (manufactured by Alpha Innotech Corporation),the HSP70 gene the expression level was measured. On this occasion, avalue obtained by being corrected with the expression level of theinternal control gene was regarded as the expression level of HSP70gene.

The results are shown in FIG. 2. In FIG. 2, the HSP70 mRNA expressioninducing activity of the samples 1, 3, and 4 was expressed as a ratio(%) to that of the control. As compared with the control, the samples 1,3, and 4 exhibited an about 1.2- to 1.5-fold increased HSP70 mRNAexpression (samples 1 and 4, **p<0.01 vs. control; sample 3, *p<0.05 vs.control). From this, it became apparent that hydroxymethylfurfural, andthe product obtained by heat-treating an asparagus stem with hot water,and the product obtained by heat-treating an asparagus stem with hotwater and enzyme treatment by this Example had the HSP70 inducingactivity at an mRNA expression level.

Example 5

(Evaluation of HSP70 Protein Expression Inducing Activity)

The HSP70 inducing activity of the samples 1, 3, and 4 which were thesame ones as used in Example 4 was evaluated by measuring an HSP70protein expression level.

In the same manner as described in Example 4, HeLa cells that had beensuspended in DMEM (added with 10% FBS) were seeded in a 12-well plate(100,000 cells/mL/well). On the following day, the medium was replacedwith fresh DMEM (0.9 mL); and 0.1 mL of each of the samples was added,which samples had been each prepared with ion-exchanged water so as tohave a final concentration of 1 mg/mL (with regard to the samples 3 and4, a final concentration of 1 mg/mL in terms of a product obtained byheat-treating an asparagus stem with hot water and a product obtained byheat-treating an asparagus stem with hot water and enzyme treatmentderived from the solid content of asparagus stem). To a control,ion-exchanged water 0.1 mL was added. After culturing for 24 hours, aculture supernatant was removed and cells were washed with PBS(−)(phosphate buffered physiological saline, pH 7.2). A part of the cellswere then scraped off with a cell scraper, collected into a 1.5 mLsample tube, and subjected to HSP70 protein quantification and totalprotein quantification.

The quantification of HSP70 protein was carried out using HSP70 ELISAkit (product name) (manufactured by Enzo); and the quantification oftotal protein was carried out using Micro BCA Protein Assay Reagent kit(product name) (manufactured by PIERCE Biotechnology). With regard tothe remaining cells, an effect on cell proliferation was evaluated by3-(4,5-dimethyl thial-2-yl)-2,5-diphenyltetrazalium bromide (MTT)method. Subsequently, a value corrected with the amount of totalproteins and the number of viable cells was regarded as the amount ofHSP70 protein.

The results are shown in FIG. 3. In FIG. 3, the HSP70 protein expressioninducing activity of the samples 1, 3, and 4 was expressed as a ratio(%) to that of the control. As compared with the control, the samples 1,3, and 4 exhibited an about 1.3-fold increased HSP70 protein expression(*p<0.05 vs. control). From this, it became apparent thathydroxymethylfurfural, the product obtained by heat-treating anasparagus stem with hot water, and the product obtained by heat-treatingan asparagus stem with hot water and enzyme treatment by this Examplehad the HSP70 inducing activity at a protein expression level.

From the above, it was demonstrated that hydroxymethylfurfural, theproduct obtained by heat-treating an asparagus stem with hot water, andthe product obtained by heat-treating an asparagus stem with hot waterand enzyme treatment by this Example had an excellent HSP70 inducingactivity.

Example 6

(Evaluation of Anti-Stress Effect in Mouse Model of Sleep Seprivation)

The anti-stress effect of the product obtained by heat-treating anasparagus stem with hot water which was obtained in Example 4 (the“sample 3” in Example 4), was evaluated using a mouse model of sleepdeprivation.

Thirty two male Slc:ddY mice at 6 weeks of age (manufactured by CLEAJapan, Inc.) were divided into 4 groups (8 mice in each group). Each ofthe groups was designated as a normal group, a control group, a groupwith a low dose of the product obtained by heat-treating an asparagusstem with hot water (hereinafter, referred to as a low dose group), anda group with a high dose of the product obtained by heat-treating anasparagus stem with hot water (hereinafter, referred to as a high dosegroup). Starting seven days before mice were put under the stress ofsleep deprivation, a powder containing the product obtained byheat-treating an asparagus stem with hot water was added to regular mashfeed (product name: CE-2, manufactured by CLEA Japan, Inc.) and fedeveryday to mice in the low dose group at a dose of 200 mg/kg (in termsof the product obtained by heat-treating an asparagus stem with hotwater derived from the solid content of asparagus stem) and to mice inthe high dose group at a dose of 1000 mg/kg (in terms of the same). Thenormal group and control group were fed the regular mash feed. In thecontrol group, low dose group, and high dose group, mice were immersedin water for 12 hours (8:00 to 20:00) per day to apply the stress ofsleep deprivation over 3 days. To the normal group, the stress of sleepdeprivation was not applied.

The anti-stress effect was evaluated in mice on the day following thelast load of the stress of sleep deprivation by measuring (1) lipidperoxide levels in the blood serum (a ratio of the amount of lipidperoxide (LPO) to the amount of triglyceride (TG) in the blood serum(LPO/TG)) as an oxidative stress index, (2) measuring the bloodconcentration of corticosterone which is known as a stress hormone, and(3) evaluating the incidence rate of mice with hair loss.

FIG. 4 shows the result of measurement of the lipid peroxide level inthe blood serum. In the figure, a higher value of LPO/TG indicates astate of a higher oxidative stress in the blood. The value of LPO/TG washigh in the control group whereas the value of LPO/TG decreased to thealmost same level as the normal group in the low dose group and highdose group (*p<0.05 vs. control). From this, it was shown that when theproduct obtained by heat-treating an asparagus stem with hot water bythis Example was taken by mice with a state where the oxidative stressin the blood was high because of load of the stress of sleepdeprivation, the oxidative stress in the blood was reduced to the levelat which the stress of sleep deprivation was not applied.

FIG. 5 shows the result of measurement of the corticosteroneconcentration in the blood. In the figure, a higher value ofcorticosterone concentration indicates a higher stress state. The valueof corticosterone concentration was high in the control group whereasthe value of corticosterone concentration decreased to the almost samelevel as the normal group in the low dose group and the value ofcorticosterone concentration was reduced to the same or lower level thanthe normal group in the high dose group (**p<0.01 vs. control, *p<0.05vs. control). From this, it was shown that when the product obtained byheat-treating an asparagus stem with hot water by this Example was takenby mice that were in a higher stress state because of load of the stressof sleep deprivation, the higher stress state was reduced to the levelat which the stress of sleep deprivation was not applied and, in thehigh dose group, reduced further to the same or lower level than thestress of sleep deprivation was not applied.

FIG. 6 shows the incidence rate of mice with hair loss. In the figure, ahigher incidence rate of hair loss indicates a higher stress state. Theincidence rate of hair loss in the normal group, control group, low dosegroup, and high dose group was 0%, 75.0%, 37.5%, and 12.5%,respectively; and the incidence rate of hair loss was lower in the lowdose group and high dose group, as compared with that of the controlgroup. From this, it was shown that when the product obtained byheat-treating an asparagus stem with hot water by this Example was takenby mice that were in a higher stress state because of load of the stressof sleep deprivation, the higher stress state was reduced.

From the above, it was demonstrated that the product obtained byheat-treating an asparagus stem with hot water containing thehydroxymethylfurfural derivative by this Example had an excellentanti-stress effect.

Example 7

(Evaluation of HSP70 Protein Expression Inducing Activity in a MouseModel of Sleep Deprivation)

Using the mice used in Example 6, the HSP70 inducing activity of theproduct obtained by heat-treating an asparagus stem with hot water whichwas obtained in Example 4 (the “sample 3” in Example 4) was evaluated bymeasuring the HSP70 protein expression level in the stomach, liver, andkidney.

On the last day of the study in Example 6, the mice in each of thegroups were sacrificed; and the stomach, liver, and kidney were eachharvested. Each of the organs (50 mg) was placed in a 1.5 mL sampletube; and 500 μL of the extraction reagent of HSP70 ELISA kit (productname) (manufactured by Enzo) added with Protease inhibitor cocktail(product name) (manufactured by Sigma) at 0.2% (v/v) was added thereto.Each of the organ was then mashed on ice using a pestle rod andcentrifuged at 4° C., at 1,500 rpm for 30 minutes, and the supernatantwas collected. This supernatant was subj ected to the quantification ofHSP70 protein and the quantification of total proteins.

In the same manner as described in Example 5, the quantification ofHSP70 protein was carried out using HSP70 ELISA kit (product name)(manufactured by Enzo) and the quantification of total proteins wascarried out using Micro BCA Protein Assay Reagent kit (product name)(manufactured by PIERCE Biotechnology). A value corrected with theamount of total proteins was regarded as the amount of HSP70 protein.

FIGS. 7 to 9 show the expression level of the HSP70 protein in thestomach, liver, and kidney. In FIGS. 7 to 9, the HSP70 proteinexpression inducing activity of the control group, low dose group, andhigh dose group was expressed as a ratio (%) to that of the normalgroup. In the stomach (FIG. 7) and liver (FIG. 8), the expression levelof HSP70 protein decreased in the control group as compared with thenormal group; whereas the HSP70 protein expression increased in the lowdose group and high dose group to the same or higher level than that inthe normal group (*p<0.05 vs. control). In the kidney (FIG. 9), theHSP70 protein expression increased in the low dose group and high dosegroup, as compared with the control group (**p<0.01 vs. control).

From the above, it became apparent that the product obtained byheat-treating an asparagus stem with hot water containing thehydroxymethylfurfural derivative by this Example had, even whenadministrated to animals, an excellent HSP70 inducing activity at aprotein expression level. Further, it was suggested that one of themechanisms of action of the anti-stress effect shown in Example 6 wasthe HSP70 expression inducing activity of the product obtained byheat-treating an asparagus stem with hot water containing thehydroxymethylfurfural derivative by this Example.

Example 8

(Evaluation of HSP70 mRNA Expression Inducing Activity in Human)

Using the product obtained by heat-treating an asparagus stem with hotwater which was obtained in Example 4 (the “sample 3” in Example 4), anHSP70 inducing activity in human white blood cells was evaluated bymeasuring an HSP70 mRNA expression level.

Three volunteers who voluntarily expressed intent to participate wereemployed as subjects (hereinafter, referred to as subject 1, subject 2,and subject 3). A powder containing product obtained by heat-treating anasparagus stem with hot water was taken twice a day (morning andevening) for 3 days, by the subject 1 at a dose of 200 mg/day, thesubject 2 at a dose of 400 mg/day, and the subject 3 at a dose of 800mg/day, (in 200 mg (subject 1), 400 mg (subject 2), and 800 mg (subject3) of such a powder of the product obtained by heat-treating anasparagus stem with hot water, 98 mg, 197 mg, and 394 mg of the productobtained by heat-treating an asparagus stem with hot water derived fromthe solid content of asparagus stem were contained, respectively).

Before the start of the intake and on the last day of the intake, theblood was drawn and the expression level of HSP70 mRNA in white bloodcells was measured. The blood 1 mL was mixed with 10 mL of ACK buffersolution (0.15 M ammonium chloride, 1.0 mM potassium bicarbonate, 0.1 mMEDTA-2Na, pH 7.2) at 37° C., and kept at 37° C., for 10 minutes. Theresultant was then centrifuged at 3,000 rpm for 5 minutes and thesupernatant was removed. The precipitated white blood cells were againadded with 10 mL of ACK buffer solution to be suspended. The sameprocedure was repeated three times; and Trizol reagent (product name)(Life Technologies) 1.5 mL was added to the precipitated white bloodcells to extract total RNA. Procedures subsequent to this, including aPCR reaction, were carried out in the same manner as described inExample 4 (the evaluation in HeLa cells); and the expression amount ofHSP70 mRNA was evaluated.

The results are shown in FIG. 10. In FIG. 10, a ratio (%) of theexpression level of HSP70 mRNA in the white blood cells after thecompletion of the intake to that before the start of the intake ispresented. The HSP70 mRNA expression in the white blood cells after thecompletion of the intake increased about 2.5 to 3.5 fold in a fashiondependent on the dose of the product obtained by heat-treating anasparagus stem with hot water, as compared with that before the start ofthe intake.

From the above, it became apparent that the product obtained byheat-treating an asparagus stem with hot water containing thehydroxymethylfurfural derivative by this Example had, even whenadministrated in human, an excellent HSP70 inducing activity at an mRNAexpression level.

Example 9

(Clinical Evaluation of Autonomic Nerve Regulatory Effect of a ProductObtained by Heat-Treating an Asparagus Stem with Hot Water)

Using the product obtained by heat-treating an asparagus stem with hotwater which was obtained in Example 4 (the “sample 3” in Example 4), anautonomic nerve regulatory effect in human was evaluated.

Thirty volunteers who voluntarily expressed intent to participate wereemployed as subjects to carry out a randomized placebo controlled doubleblind study. The subjects were allocated, by using a lottery, to aplacebo group (hereinafter, referred to as P group) 15 subjects, or agroup with the product obtained by heat-treating an asparagus stem withhot water (hereinafter, referred to as A group) 15 subjects. Over fourweeks during the study period, the subjects in the P group took anexcipient (product name: Pinedex, manufactured by Matsutani ChemicalIndustry Co., Ltd.) (400 mg/day) whereas the subjects in the A grouptook a powder containing the product obtained by heat-treating anasparagus stem with hot water (400 mg/day) twice a day (morning andevening) everyday (in 400 mg of such a powder of the product obtained byheat-treating an asparagus stem with hot water, the product obtained byheat-treating an asparagus stem with hot water derived from the solidcontent of asparagus stem accounted for 197 mg and the remaining 203 mgwas the excipient (same as above)).

Before the start of the study and on the last day of the study, anautonomic nervous balance and autonomic nervous activity were evaluatedusing an acceleration pulse wave inspection system (product name; Pulseanalyzer plus TAS-9, manufactured by YKC Corporation). Such a system isa system in which acceleration pulse waves are measured at the fingertipto thereby detect subtle changes in the heart rate (Heart RateVariability: HRV) and evaluate autonomic nerve functions. HRV isexpressed as clinical consequences for various influences that theautonomic nerve brings about to the heart rate. The autonomic nervousbalance was evaluated by plotting an index for sympathetic nerveactivity (Low Frequency: LF) on the X axis and an index forparasympathetic nerve activity (High Frequency: HF) on the Y axis, whichactivities were given by such a system, to prepare a two-dimensionalgraph, and using a distance between the point at which the autonomicnervous balance was best and the point at a measured value on such agraph. Meanwhile, with regard to the autonomic nervous activity,numerical values representing the activity of the autonomic nerve thatwas given by such a system (the numerical values being calculated usingLF, HF, and the like by such a system) were used.

FIG. 11 shows changes in the autonomic nervous balance. In the figure, acloser numerical value to zero indicates a better balance of theautonomic nerve. In the P group (the placebo group), the balance of theautonomic nerve on the last day of the study worsened, as compared withthat before the start of the study. On the other hand, in the A group(the group with the product obtained by heat-treating an asparagus stemwith hot water), the balance of the autonomic nerve on the last day ofthe study significantly improved, as compared with that before the startof the study (*p<0.05 vs. before the start of the study, p<0.01 vs. theplacebo group). From this, it was demonstrated that, by taking theproduct obtained by heat-treating an asparagus stem with hot water bythis Example, the balance of the autonomic nerve improved.

FIG. 12 shows changes in the autonomic nervous activity. In the figure,a higher numerical value indicates a higher autonomic nervous activity.In the P group (the placebo group), a decreased autonomic nervousactivity was found on the last day of the study, as compared with thatbefore the start of the study. On the other hand, in the A group (thegroup with the product obtained by heat-treating an asparagus stem withhot water), an elevated autonomic nervous activity was found on the lastday of the study, as compared with that before the start of the study.From this, it was demonstrated that, by taking the product obtained byheat-treating an asparagus stem with hot water by this Example, theautonomic nervous activity improved.

From the above, it was demonstrated that the product obtained byheat-treating an asparagus stem with hot water containing thehydroxymethylfurfural derivative by this Example had an excellentautonomic nerve regulatory effect.

Example 10

(Evaluation of HSP70 mRNA expression inducing activity of a productobtained by heat-treating an asparagus stem with hot water and enzymetreatment in human and clinical evaluation of autonomic nerve regulatoryeffect thereof)

Using the obtained capsule filled with the product obtained byheat-treating an asparagus stem with hot water and enzyme treatment,evaluation of an HSP70 mRNA expression inducing activity in human andclinical evaluation of an autonomic nerve regulatory effect were carriedout as described below.

(Method of pProducing a Capsule Filled with a Product Obtained byHeat-Treating an Asparagus Stem with Hot Water and Enzyme Treatment)

A method for producing a capsule filled with a product obtained byheat-treating an asparagus stem with hot water and enzyme treatment forhuman intake is shown below. To green asparagus stems (fresh weight 130kg), water 170 L was added; and the mixture was, for the purpose of heattreatment with hot water, subjected to heat sterilization (100° C., 45minutes). After allowed to cool to 45° C., the resultant was added with3.0 kg of enzymes (Sumizyme C and Sumizyme MC; manufactured by YakultPharmaceutical Industry Co., Ltd.) and stirred at 45° C., for 24 hours.The enzymes were then deactivated (100° C., 20 minutes); andcentrifugation was carried out. The resultant was concentrated by anevaporator, added with 9.0 kg of excipient (product name: Pinedex,manufactured by Matsutani Chemical Industry Co., Ltd.), and autoclaved(121° C., 45 minutes). Subsequently, by spray drying, a powdercontaining the product obtained by heat-treating an asparagus stem withhot water and enzyme treatment 16.0 kg (in this, the product obtained byheat-treating an asparagus stem with hot water and enzyme treatmentderived from the solid content of asparagus stem accounted for 7.0 kgand the excipient accounted for 9.0 kg) was obtained. This powdercontaining the product obtained by heat-treating an asparagus stem withhot water and enzyme treatment 8.50 kg was mixed with an anticakingagent (product name: Calcium stearate, manufactured by Sun AceCorporation) 1.86 kg and cellulose (product name: Ceolus, manufacturedby Asahi Kasei Corp.) 8.20 kg to prepare a powder for capsulescontaining 20% the product obtained by heat-treating an asparagus stemwith hot water and enzyme treatment derived from the solid content ofasparagus stem. One obtained by filling this powder for capsules in No.1capsule at 280 mg per capsule was used as to a capsule filled with theproduct obtained by heat-treating an asparagus stem with hot water andenzyme treatment.

(Method of Evaluation)

Twenty volunteers who voluntarily expressed intent to participate wereemployed as subjects; and a short term randomized placebo controlleddouble blind study was carried out at a low dose. The subjects wererandomly allocated to a placebo group (hereinafter, referred to as Pgroup) 10 subjects or a group with a product obtained by heat-treatingan asparagus stem with hot water and enzyme treatment (hereinafter,referred to as E group) 10 subjects. Over one week during the studyperiod, the subjects in the P group took a placebo capsule (a total of840 mg of mixture of product name: Pinedex (manufactured by MatsutaniChemical Industry Co., Ltd.) 699.9 mg and product name: Malt extract(Oriental Kogyo) 140.1 mg (3 capsules)/day) everyday whereas thesubjects in the E group took the capsule filled with the productobtained by heat-treating an asparagus stem with hot water and enzymetreatment (840 mg (3 capsules)/day) after dinner every day (in 840 mg ofsuch a capsule filled with product obtained by heat-treating anasparagus stem with hot water and enzyme treatment, the product obtainedby heat-treating an asparagus stem with hot water and enzyme treatmentderived from the solid content of asparagus stem accounted for 168 mgand the remaining 672 mg was Pinedex, calcium stearate, and Ceolus). Theexpression amount of HSP70 mRNA in white blood cells, autonomic nervousbalance, and autonomic nervous activity are employed as endpoints.

(HSP70 mRNA expression inducing activity evaluation)

First, the expression level of HSP70 mRNA in white blood cells wasmeasured. Before the start of the study and on the last day of thestudy, the blood was drawn; and total RNA was extracted from 400 μL ofthe blood using an RNA extraction kit (product name: Nucleo Spin RNABlood, manufactured by Takara Bio Inc.). The method of cDNA synthesisand PCR conformed to the method described in Example 8.

The results are shown in FIG. 13. In FIG. 13, a ratio (%) of theexpression level of HSP70 mRNA in the white blood cells after thecompletion of the intake to that before the start of the intake ispresented. The expression level of HSP70 mRNA in the P group (theplacebo group) was, on the basis of the average value, 175% of thatbefore the start of the intake. On the other hand, the expression levelof HSP70 mRNA in the E group (the group with the product obtained byheat-treating an asparagus stem with hot water and enzyme treatment)was, on the basis of the average value, 278% of that before the start ofthe intake; and the expression increased (*p=0.098 vs. the P group).From this, it was demonstrated that, by taking the product obtained byheat-treating an asparagus stem with hot water and enzyme treatment bythis Example, the expression amount of HSP70 mRNA increased markedly.

(Clinical Evaluation on Autonomic Nerve Regulatory Effect)

Next, the autonomic nervous balance and autonomic nervous activity wereevaluated using an acceleration pulse wave inspection system (productname; Pulse analyzer plus TAS-9, manufactured by YKC Corporation) beforethe start of the study and on the last day of the study. The details ofmeasurement were the same as described in Example 9.

FIG. 14 shows changes in the autonomic nervous balance. In the P group(the placebo group), the autonomic nervous balance on the last day ofthe study significantly worsened, as compared with that before the startof the study (*p<0.05 vs. before the start of the study). On the otherhand, in the E group (the group with the product obtained byheat-treating an asparagus stem with hot water and enzyme treatment),the balance of the autonomic nerve on the last day of the studyimproved, as compared with that before the start of the study. Fromthis, it was demonstrated that, by taking the product obtained byheat-treating an asparagus stem with hot water and enzyme treatment bythis Example, the balance of the autonomic nerve improved.

FIG. 15 shows changes in the autonomic nervous activity. In the P group(the placebo group), the autonomic nervous activity on the last day ofthe study significantly worsened, as compared with that before the startof the study (**p<0.01 vs. before the start of the study). On the otherhand, in the E group (the group with the product obtained byheat-treating an asparagus stem with hot water and enzyme treatment),the autonomic nervous activity on the last day of the study improved, ascompared with that before the start of the study. From this, it wasdemonstrated that, by taking the product obtained by heat-treating anasparagus stem with hot water and enzyme treatment by this Example,deterioration of the autonomic nervous activity was prevented.

As described above, according to the present disclosure, a novelhydroxymethylfurfural derivative, a highly effective pharmaceutical, HSPinducer, anti-stress agent, and autonomic nerve regulator can beprovided. In addition, foods and drinks having an excellent HSP inducingactivity, anti-stress effect, and autonomic nerve regulatory effect canbe provided. Further, a method of producing a hydroxymethylfurfuralderivative that can reduce the cost and is simple and convenient can beprovided.

It is to be noted that various embodiments and modifications arefeasible in the present disclosure without departing from the broadspirit and scope of the present disclosure. Further, the above-mentionedembodiments are intended to illustrate the present disclosure and arenot intended to limit the scope of the present disclosure. That is, thescope of the present disclosure is indicated by the claims rather thanby the embodiments. And various modifications which come within theclaims and within the meaning of invention equivalent to the claims aredeemed to be within the scope of the present disclosure.

We claim:
 1. A method for induction of a heat shock protein,anti-stress, or regulation of an autonomic nerve comprisingadministering a mammalian subject in need thereof once or twice daily acomposition comprising an effective amount of hydroxymethylfurfuralderivative of the following formula:

wherein R is selected from the group consisting of formula (I):

(II): HOOCCH₂COCO—, (III): HOOCCH₂CH₂COCO—, and (IV): a hydrogen atom,at a dosage of 50 mg to 2000 mg per day, wherein the dosage is in a formselected from the group consisting of granules, particulates, tablets,capsules, suspensions, aqueous solutions, emulsions, and powders.
 2. Themethod according to claim 1 wherein the hydroxymethylfurfural derivativeis obtained by heat-treating an asparagus stem with hot water and anenzyme treatment, wherein the enzyme treatment is carried out by addingat least one enzyme selected from among a group consisting of cellulase,hemicellulase, pectinase, amylase and pullulanase; and treating at atemperature of 30 to 60° C. , for 1 to 72 hours.
 3. The method of claim1 wherein the composition comprises powders prepared by a methodcomprising: heat-treating an asparagus stem with hot water to obtain aheat-treated mixture for about 20 to 180 minutes; cooling; filtering;concentrating; adding a pharmaceutically acceptable excipient; anddrying.
 4. The method of claim 1 wherein the dosage is in a formselected from the group consisting of tablets, capsules, suspensions,aqueous solutions, emulsions, and powders.
 5. A method for induction ofa heat shock protein, anti-stress, or regulation of an autonomic nervecomprising administering a mammalian subject in need thereof a productobtained by heat-treating an asparagus stem with hot water and an enzymetreatment, wherein the enzyme treatment is carried out by adding atleast one enzyme selected from among a group consisting of cellulase,hemicellulase, pectinase, amylase and pullulanase; and treating at atemperature of 30 to 60° C., for 1 to 72 hours.
 6. The method of claim 5wherein the product is in a form selected from the group consisting ofgranules, particulates, tablets, capsules, suspensions, aqueoussolutions, emulsions, and powders, and administered once or twice dailyat a dosage of 50 mg to 2000 mg per day.
 7. The method of claim 6wherein the product is in a form selected from the group consisting oftablets, capsules, suspensions, aqueous solutions, emulsions, andpowders.
 8. A method for induction of a heat shock protein, anti-stressor regulation of an autonomic nerve comprising: administering amammalian subject in need thereof a composition comprising an effectiveamount of hydroxymethylfurfural derivative of the following formula:

wherein R is selected from the group consisting of formula (I):

(II): HOOCCH₂COCO—, (III): HOOCCH₂CH₂COCO—, and (IV): a hydrogen atom,and evaluating heat shock protein activity, stress, autonomic nervousbalance, or autonomic nervous activity of the mammalian subject.
 9. Themethod of claim 8 wherein the evaluating step is conducted at least oncebefore the administrating step and at least once after theadministrating step.
 10. The method of claim 8 wherein theadministrating step comprises administrating the mammalian subject thecomposition 50 mg to 2000 mg/day.
 11. The method of claim 8 wherein theadministrating step comprises administrating the mammalian subject thecomposition once or twice daily.
 12. The method of claim 8 wherein thecomposition is in a dosage form selected from the group consisting ofgranules, particulates, tablets, capsules, suspensions, aqueoussolutions, emulsions, and powders.
 13. The method according to claim 8wherein the composition is in a dosage form selected from the groupconsisting of tablets, capsules, suspensions, aqueous solutions,emulsions, and powders.
 14. The method according to claim 8 wherein thehydroxymethylfurfural derivative is obtained by heat-treating anasparagus stem with hot water and an enzyme treatment, wherein theenzyme treatment is carried out by adding at least one enzyme selectedfrom among a group consisting of cellulase, hemicellulase, pectinase,amylase and pullulanase; and treating at a temperature of 30 to 60° C. ,for 1 to 72 hours.
 15. The method of claim 8 wherein the compositioncomprises powders prepared by a method comprising: heat-treating anasparagus stem with hot water to obtain a heat-treated mixture for about20 to 180 minutes; cooling; filtering; concentrating; adding anpharmaceutically acceptable excipient; and drying.